Part:BBa_K5425005
Contents
Introduction
This circuit is designed to fine-tune the parameters of various components of part BBa_K5425004. By measuring the levels of the fluorophore, we can indirectly relate its expression to the gene of interest, which we aim to produce in an oscillatory manner. Specifically, we plan to express a cell cycle arrest gene, resulting in periodic oscillations in cell growth.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2493
Illegal AgeI site found at 3539 - 1000COMPATIBLE WITH RFC[1000]
Modeling and Characterization
Fig: Oscillation in GFP Expression modeled through ODE.
Cloning and expression
All the sequences were sourced either from NCBI or the Parts Registry and then assembled in silico using SnapGene. For cloning, we used the part BBa K5425004 which was earlier cloned by us. These segments were synthesized and assembled using scarless NEBuilder® HiFi DNA Assembly. Finally, the assembled parts were transformed into BL21 competent E. coli.
Discussion
Fluorescence was initially not prominent after inducing the circuit, but we observed a significant increase upon treatment with aTc. We believe the main reason for not detecting oscillations is the use of a long-lived GFP variant. Oscillations can only be effectively captured if the half-life of the GFP molecule is shorter than the oscillation time period, allowing the fluorescence to reflect the dynamic changes accurately.
To get insights over the experiments and design please visit :- https://2024.igem.wiki/iiser-kolkata/
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